Antimicrobial mitochondrial reactive oxygen species induction by lung epithelial immunometabolic modulation.

Pneumonia is a worldwide threat, making discovery of novel means to combat lower respiratory tract infection an urgent need. Manipulating the lungs' intrinsic host defenses by therapeutic delivery of certain pathogen-associated molecular patterns protects mice against pneumonia in a reactive oxygen species (ROS)-dependent manner. Here we show that antimicrobial ROS are induced from lung epithelial cells by interactions of CpG oligodeoxynucleotides (ODN) with mitochondrial voltage-dependent anion channel 1 (VDAC1). The ODN-VDAC1 interaction alters cellular ATP/ADP/AMP localization, increases delivery of electrons to the electron transport chain (ETC), increases mitochondrial membrane potential (ΔΨm), differentially modulates ETC complex activities and consequently results in leak of electrons from ETC complex III and superoxide formation. The ODN-induced mitochondrial ROS yield protective antibacterial effects. Together, these studies identify a therapeutic metabolic manipulation strategy to broadly protect against pneumonia without reliance on antibiotics.

Meta-omics profiling of the gut-lung axis illuminates metabolic networks and host-microbial interactions associated with elevated lung elastance in a murine model of obese allergic asthma.

Obesity and associated changes to the gut microbiome worsen airway inflammation and hyperresponsiveness in asthma. Obesogenic host-microbial metabolomes have altered production of metabolites that may influence lung function and inflammatory responses in asthma. To understand the interplay of the gut microbiome, metabolism, and host inflammation in obesity-associated asthma, we used a multi-omics approach to profile the gut-lung axis in the setting of allergic airway disease and diet-induced obesity. We evaluated an immunomodulator, nitro-oleic acid (NO2-OA), as a host- and microbial-targeted treatment intervention for obesity-associated allergic asthma. Allergic airway disease was induced using house dust mite and cholera toxin adjuvant in C57BL6/J mice with diet-induced obesity to model obesity-associated asthma. Lung function was measured by flexiVent following a week of NO2-OA treatment and allergen challenge. 16S rRNA gene (from DNA, taxa presence) and 16S rRNA (from RNA, taxa activity) sequencing, metabolomics, and host gene expression were paired with a Treatment-Measured-Response model as a data integration framework for identifying latent/hidden relationships with linear regression among variables identified from high-dimensional meta-omics datasets. Targeting both the host and gut microbiota, NO2-OA attenuated airway inflammation, improved lung elastance, and modified the gut microbiome. Meta-omics data integration and modeling determined that gut-associated inflammation, metabolites, and functionally active gut microbiota were linked to lung function outcomes. Using Treatment-Measured-Response modeling and meta-omics profiling of the gut-lung axis, we uncovered a previously hidden network of interactions between gut levels of amino acid metabolites involved in elastin and collagen synthesis, gut microbiota, NO2-OA, and lung elastance. Further targeted metabolomics analyses revealed that obese mice with allergic airway disease had higher levels of proline and hydroxyproline in the lungs. NO2-OA treatment reduced proline biosynthesis by downregulation of pyrroline-5-carboxylate reductase 1 (PYCR1) expression. These findings are relevant to human disease: adults with mild-moderate asthma and BMI ≥ 25 had higher plasma hydroxyproline levels. Our results suggest that changes to structural proteins in the lung airways and parenchyma may contribute to heightened lung elastance and serve as a potential therapeutic target for obese allergic asthma.

Antimicrobial mitochondrial reactive oxygen species induction by lung epithelial metabolic reprogramming.

Pneumonia is a worldwide threat, making discovery of novel means to combat lower respiratory tract infections an urgent need. We have previously shown that manipulating the lungs' intrinsic host defenses by therapeutic delivery of a unique dyad of pathogen-associated molecular patterns protects mice against pneumonia in a reactive oxygen species (ROS)-dependent manner. Here we show that antimicrobial ROS are induced from lung epithelial cells by interactions of CpG oligodeoxynucleotides (ODNs) with mitochondrial voltage-dependent anion channel 1 (VDAC1) without dependence on Toll-like receptor 9 (TLR9). The ODN-VDAC1 interaction alters cellular ATP/ADP/AMP localization, increases delivery of electrons to the electron transport chain (ETC), enhances mitochondrial membrane potential (Δ Ψm ), and differentially modulates ETC complex activities. These combined effects promote leak of electrons from ETC complex III, resulting in superoxide formation. The ODN-induced mitochondrial ROS yield protective antibacterial effects. Together, these studies identify a therapeutic metabolic manipulation strategy that has the potential to broadly protect patients against pneumonia during periods of peak vulnerability without reliance on currently available antibiotics. Author Summary: Pneumonia is a major cause of death worldwide. Increasing antibiotic resistance and expanding immunocompromised populations continue to enhance the clinical urgency to find new strategies to prevent and treat pneumonia. We have identified a novel inhaled therapeutic that stimulates lung epithelial defenses to protect mice against pneumonia in a manner that depends on production of reactive oxygen species (ROS). Here, we report that the induction of protective ROS from lung epithelial mitochondria occurs following the interaction of one component of the treatment, an oligodeoxynucleotide, with the mitochondrial voltage-dependent anion channel 1. This interaction alters energy transfer between the mitochondria and the cytosol, resulting in metabolic reprogramming that drives more electrons into the electron transport chain, then causes electrons to leak from the electron transport chain to form protective ROS. While antioxidant therapies are endorsed in many other disease states, we present here an example of therapeutic induction of ROS that is associated with broad protection against pneumonia without reliance on administration of antibiotics.

Correction: Epigenetic loss of AOX1 expression via EZH2 leads to metabolic deregulations and promotes bladder cancer progression.

After publication of this Article, the Authors noticed errors in some of the Figures. In Figures 2e, 2f-g, 4a, 4j, 5a and 6b, unmatched ß-actin was inadvertently used as loading control for the immunoblots. These have been corrected using repeat data from a similar set of samples and the revised Figures containing matched ß-actin and their respective quantification data are included below. In Figure 7a, the same image was inadvertently used to represent tumors 3 and 5 in the control group. This error has been corrected using original images of tumors 3 and 5 in the control group. Additional corrections have been made in the Article and Figure legends to enhance the clarity of the description. NAD was replaced by NADP. NAD/NADP was replaced by NADP/NADPH. The description of the antibody source and dilution for the antigens PFKFB4 (Abcam, 1:1000), G6PD, and HK1 (Cell Signaling, 1:1,000) have been included in the Methods section for Western Blot. The legend for Figure 4e and 4j has been updated. The HTML and PDF versions of this Article have been corrected. The scientific conclusions of this paper have not been affected.

Epigenetic loss of AOX1 expression via EZH2 leads to metabolic deregulations and promotes bladder cancer progression.

Advanced Bladder Cancer (BLCA) remains a clinical challenge that lacks effective therapeutic measures. Here, we show that distinct, stage-wise metabolic alterations in BLCA are associated with the loss of function of aldehyde oxidase (AOX1). AOX1 associated metabolites have a high predictive value for advanced BLCA and our findings demonstrate that AOX1 is epigenetically silenced during BLCA progression by the methyltransferase activity of EZH2. Knockdown (KD) of AOX1 in normal bladder epithelial cells re-wires the tryptophan-kynurenine pathway resulting in elevated NADP levels which may increase metabolic flux through the pentose phosphate (PPP) pathway, enabling increased nucleotide synthesis, and promoting cell invasion. Inhibition of NADP synthesis rescues the metabolic effects of AOX1 KD. Ectopic AOX1 expression decreases NADP production, PPP flux and nucleotide synthesis, while decreasing invasion in cell line models and suppressing growth in tumor xenografts. Further gain and loss of AOX1 confirm the EZH2-dependent activation, metabolic deregulation, and tumor growth in BLCA. Our findings highlight the therapeutic potential of AOX1 and provide a basis for the development of prognostic markers for advanced BLCA.

Correction: Expression of ganglioside GD2, reprogram the lipid metabolism and EMT phenotype in bladder cancer.

[This corrects the article DOI: 10.18632/oncotarget.21038.].

Measurement of methylated metabolites using Liquid Chromatography-Mass Spectrometry and its biological application.

Methylation aberrations play an important role in many metabolic disorders including cancer. Methylated metabolites are direct indicators of metabolic aberrations, and currently, there is no Liquid chromatography - Mass spectrometry (LC-MS) based method available to cover all classes of methylated metabolites at low detection limits. In this study, we have developed a method for the detection of methylated metabolites, and it's biological application. In this approach, we used a HILIC based HPLC with MS to measure methylated organic acids, amino acids, and nucleotides. These metabolites were separated from each other by their hydrophobic interactions and analyzed by targeted metabolomics of single reaction monitoring by positive and negative mode of electrospray ionization. These metabolites were quantified, and the interday reproducibility was <10% relative standard deviation. Furthermore, by applying this method, we identified high levels of methylated metabolites in bladder cancer cell lines compared to benign cells. In vitro treatment of cancer cells with methylation inhibitor, 5- aza-2'-deoxycytidine showed a decrease in these methylated metabolites. This data indicates that HPLC analysis using this HILIC based method could be a powerful tool for measuring methylated metabolites in biological specimens. This method is rapid, sensitive, selective, and precise to measure methylated metabolites.

DNA methylation patterns in bladder tumors of African American patients point to distinct alterations in xenobiotic metabolism.

Racial/ethnic disparities have a significant impact on bladder cancer outcomes with African American patients demonstrating inferior survival over European-American patients. We hypothesized that epigenetic difference in methylation of tumor DNA is an underlying cause of this survival health disparity. We analyzed bladder tumors from African American and European-American patients using reduced representation bisulfite sequencing (RRBS) to annotate differentially methylated DNA regions. Liquid chromatography-mass spectrometry (LC-MS/MS) based metabolomics and flux studies were performed to examine metabolic pathways that showed significant association to the discovered DNA methylation patterns. RRBS analysis showed frequent hypermethylated CpG islands in African American patients. Further analysis showed that these hypermethylated CpG islands in patients are commonly located in the promoter regions of xenobiotic enzymes that are involved in bladder cancer progression. On follow-up, LC-MS/MS revealed accumulation of glucuronic acid, S-adenosylhomocysteine, and a decrease in S-adenosylmethionine, corroborating findings from the RRBS and mRNA expression analysis indicating increased glucuronidation and methylation capacities in African American patients. Flux analysis experiments with 13C-labeled glucose in cultured African American bladder cancer cells confirmed these findings. Collectively, our studies revealed robust differences in methylation-related metabolism and expression of enzymes regulating xenobiotic metabolism in African American patients indicate that race/ethnic differences in tumor biology may exist in bladder cancer.

Oncogenic lncRNA downregulates cancer cell antigen presentation and intrinsic tumor suppression.

How tumor cells genetically lose antigenicity and evade immune checkpoints remains largely elusive. We report that tissue-specific expression of the human long noncoding RNA LINK-A in mouse mammary glands initiates metastatic mammary gland tumors, which phenotypically resemble human triple-negative breast cancer (TNBC). LINK-A expression facilitated crosstalk between phosphatidylinositol-(3,4,5)-trisphosphate and inhibitory G-protein-coupled receptor (GPCR) pathways, attenuating protein kinase A-mediated phosphorylation of the E3 ubiquitin ligase TRIM71. Consequently, LINK-A expression enhanced K48-polyubiquitination-mediated degradation of the antigen peptide-loading complex (PLC) and intrinsic tumor suppressors Rb and p53. Treatment with LINK-A locked nucleic acids or GPCR antagonists stabilized the PLC components, Rb and p53, and sensitized mammary gland tumors to immune checkpoint blockers. Patients with programmed ccll death protein-1(PD-1) blockade-resistant TNBC exhibited elevated LINK-A levels and downregulated PLC components. Hence we demonstrate lncRNA-dependent downregulation of antigenicity and intrinsic tumor suppression, which provides the basis for developing combinational immunotherapy treatment regimens and early TNBC prevention.

Multi-omics Integration Analysis Robustly Predicts High-Grade Patient Survival and Identifies CPT1B Effect on Fatty Acid Metabolism in Bladder Cancer.

PURPOSE: The perturbation of metabolic pathways in high-grade bladder cancer has not been investigated. We aimed to identify a metabolic signature in high-grade bladder cancer by integrating unbiased metabolomics, lipidomics, and transcriptomics to predict patient survival and to discover novel therapeutic targets. EXPERIMENTAL DESIGN: We performed high-resolution liquid chromatography mass spectrometry (LC-MS) and bioinformatic analysis to determine the global metabolome and lipidome in high-grade bladder cancer. We further investigated the effects of impaired metabolic pathways using in vitro and in vivo models. RESULTS: We identified 519 differential metabolites and 19 lipids that were differentially expressed between low-grade and high-grade bladder cancer using the NIST MS metabolomics compendium and lipidblast MS/MS libraries, respectively. Pathway analysis revealed a unique set of biochemical pathways that are highly deregulated in high-grade bladder cancer. Integromics analysis identified a molecular gene signature associated with poor patient survival in bladder cancer. Low expression of CPT1B in high-grade tumors was associated with low FAO and low acyl carnitine levels in high-grade bladder cancer, which were confirmed using tissue microarrays. Ectopic expression of the CPT1B in high-grade bladder cancer cells led to reduced EMT in in vitro, and reduced cell proliferation, EMT, and metastasis in vivo. CONCLUSIONS: Our study demonstrates a novel approach for the integration of metabolomics, lipidomics, and transcriptomics data, and identifies a common gene signature associated with poor survival in patients with bladder cancer. Our data also suggest that impairment of FAO due to downregulation of CPT1B plays an important role in the progression toward high-grade bladder cancer and provide potential targets for therapeutic intervention.

Large-scale profiling of serum metabolites in African American and European American patients with bladder cancer reveals metabolic pathways associated with patient survival.

BACKGROUND: African Americans (AAs) experience a disproportionally high rate of bladder cancer (BLCA) deaths even though their incidence rates are lower than those of other patient groups. Using a metabolomics approach, this study investigated how AA BLCA may differ molecularly from European Americans (EAs) BLCA, and it examined serum samples from patients with BLCA with the aim of identifying druggable metabolic pathways in AA patients. METHODS: Targeted metabolomics was applied to measure more than 300 metabolites in serum samples from 2 independent cohorts of EA and AA patients with BLCA and healthy EA and AA controls via liquid chromatography-mass spectrometry, and this was followed by the identification of altered metabolic pathways with a focus on AA BLCA. A subset of the differential metabolites was validated via absolute quantification with the Biocrates AbsoluteIDQ p180 kit. The clinical significance of the findings was further examined in The Cancer Genomic Atlas BLCA data set. RESULTS: Fifty-three metabolites, mainly related to amino acid, lipid, and nucleotide metabolism, were identified that showed significant differences in abundance between AA and EA BLCA. For example, the levels of taurine, glutamine, glutamate, aspartate, and serine were elevated in serum samples from AA patients versus EA patients. By mapping these metabolites to genes, this study identified significant relations with regulators of metabolism such as malic enzyme 3, prolyl 3-hydroxylase 2, and lysine demethylase 2A that predicted patient survival exclusively in AA patients with BLCA. CONCLUSIONS: This metabolic profile of serum samples might be used to assess risk progression in AA BLCA. These first-in-field findings describe metabolic alterations in AA BLCA and emphasize a potential biological basis for BLCA health disparities.

Serum Metabolic Profiling Identified a Distinct Metabolic Signature in Bladder Cancer Smokers: A Key Metabolic Enzyme Associated with Patient Survival.

BACKGROUND: The current system to predict the outcome of smokers with bladder cancer is insufficient due to complex genomic and transcriptomic heterogeneities. This study aims to identify serum metabolite-associated genes related to survival in this population. METHODS: We performed LC/MS-based targeted metabolomic analysis for >300 metabolites in serum obtained from two independent cohorts of bladder cancer never smokers, smokers, healthy smokers, and healthy never smokers. A subset of differential metabolites was validated using Biocrates absoluteIDQ p180 Kit. Genes associated with differential metabolites were integrated with a publicly available cohort of The Cancer Genome Atlas (TCGA) to obtain an intersecting signature specific for bladder cancer smokers. RESULTS: Forty metabolites (FDR < 0.25) were identified to be differential between bladder cancer never smokers and smokers. Increased abundance of amino acids (tyrosine, phenylalanine, proline, serine, valine, isoleucine, glycine, and asparagine) and taurine were observed in bladder cancer smokers. Integration of differential metabolomic gene signature and transcriptomics data from TCGA cohort revealed an intersection of 17 genes that showed significant correlation with patient survival in bladder cancer smokers. Importantly, catechol-O-methyltransferase, iodotyrosine deiodinase, and tubulin tyrosine ligase showed a significant association with patient survival in publicly available bladder cancer smoker datasets and did not have any clinical association in never smokers. CONCLUSIONS: Serum metabolic profiling of bladder cancer smokers revealed dysregulated amino acid metabolism. It provides a distinct gene signature that shows a prognostic value in predicting bladder cancer smoker survival. IMPACT: Serum metabolic signature-derived genes act as a predictive tool for studying the bladder cancer progression in smokers.

Distinct Lipidomic Landscapes Associated with Clinical Stages of Urothelial Cancer of the Bladder.

BACKGROUND: The first global lipidomic profiles associated with urothelial cancer of the bladder (UCB) and its clinical stages associated with progression were identified. OBJECTIVE: To identify lipidomic signatures associated with survival and different clinical stages of UCB. DESIGN, SETTING, AND PARTICIPANTS: Pathologically confirmed 165 bladder-derived tissues (126 UCB, 39 benign adjacent or normal bladder tissues). UCB tissues included Ta (n=16), T1 (n=30), T2 (n=43), T3 (n=27), and T4 (n=9); lymphovascular invasion (LVI) positive (n=52) and negative (n=69); and lymph node status N0 (n=28), N1 (n=11), N2 (n=9), N3 (n=3), and Nx (n=75). RESULTS AND LIMITATIONS: UCB tissues have higher levels of phospholipids and fatty acids, and reduced levels of triglycerides compared with benign tissues. A total of 59 genes associated with altered lipids in UCB strongly correlate with patient survival in an UCB public dataset. Within UCB, there was a progressive decrease in the levels of phosphatidylserine (PS), phosphatidylethanolamines (PEs), and phosphocholines, whereas an increase in the levels of diacylglycerols (DGs) with tumor stage. Transcript and protein expression of phosphatidylserine synthase 1, which converts DGs to PSs, decreased progressively with tumor stage. Levels of DGs and lyso-PEs were significantly elevated in tumors with LVI and lymph node involvement, respectively. Lack of carcinoma in situ and treatment information is the limitation of our study. CONCLUSIONS: To date, this is the first study describing the global lipidomic profiles associated with UCB and identifies lipids associated with tumor stages, LVI, and lymph node status. Our data suggest that triglycerides serve as the primary energy source in UCB, while phospholipid alterations could affect membrane structure and/or signaling associated with tumor progression. PATIENT SUMMARY: Lipidomic alterations identified in this study set the stage for characterization of pathways associated with these altered lipids that, in turn, could inform the development of first-of-its-kind lipid-based noninvasive biomarkers and novel therapeutic targets for aggressive urothelial cancer of the bladder.

Expression of ganglioside GD2, reprogram the lipid metabolism and EMT phenotype in bladder cancer.

High-grade Bladder Cancer (BLCA) represents the most aggressive and treatment-resistant cancer that renders the patients with poor survival. However, only a few biomarkers have been identified for the detection and treatment of BLCA. Recent studies show that ganglioside GD2 can be used as cancer biomarker and/or therapeutic target for various cancers. Despite its potential relevance in cancer diagnosis and therapeutics, the role of GD2 is unknown in BLCA. Here, we report for the first time that high-grade BLCA tissues and cell lines have higher expression of GD2 compared to low-grade by high-resolution Mass Spectrometry. The muscle invasive UMUC3 cell line showed high GD2, mesenchymal phenotype, and cell proliferation. Besides, we have shown the cancer stem cells (CSC) property (CD44hiCD24lo) of GD2+ UMUC3 and J82 cells. Also, the evaluation of lipid metabolism in GD2+ BLCA cell lines revealed higher levels of Phosphatidylinositol (PI), Phosphatidic acid (PA), Cardiolipin (CL) and lower levels of Phosphatidylserine (PS), plasmenyl-phosphatidylethanolamines (pPE), plasmenyl-phosphocholines (pPC), sphingomyelins (SM), triglycerides (TGs) and N-Acetylneuraminic acid. These findings are significantly correlated with the tissues of BLCA patients. Based on this evidence, we propose that GD2 may be used as an effective diagnostic and therapeutic target for aggressive BLCA.

Tobacco-Specific Carcinogens Induce Hypermethylation, DNA Adducts, and DNA Damage in Bladder Cancer.

Smoking is a major risk factor for the development of bladder cancer; however, the functional consequences of the carcinogens in tobacco smoke and bladder cancer-associated metabolic alterations remain poorly defined. We assessed the metabolic profiles in bladder cancer smokers and non-smokers and identified the key alterations in their metabolism. LC/MS and bioinformatic analysis were performed to determine the metabolome associated with bladder cancer smokers and were further validated in cell line models. Smokers with bladder cancer were found to have elevated levels of methylated metabolites, polycyclic aromatic hydrocarbons, DNA adducts, and DNA damage. DNA methyltransferase 1 (DNMT1) expression was significantly higher in smokers than non-smokers with bladder cancer. An integromics approach, using multiple patient cohorts, revealed strong associations between smokers and high-grade bladder cancer. In vitro exposure to the tobacco smoke carcinogens, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and benzo[a]pyrene (BaP) led to increase in levels of methylated metabolites, DNA adducts, and extensive DNA damage in bladder cancer cells. Cotreatment of bladder cancer cells with these carcinogens and the methylation inhibitor 5-aza-2'-deoxycytidine rewired the methylated metabolites, DNA adducts, and DNA damage. These findings were confirmed through the isotopic-labeled metabolic flux analysis. Screens using smoke-associated metabolites and DNA adducts could provide robust biomarkers and improve individual risk prediction in bladder cancer smokers. Noninvasive predictive biomarkers that can stratify the risk of developing bladder cancer in smokers could aid in early detection and treatment. Cancer Prev Res; 10(10); 588-97. ©2017 AACR.